Comparative genomics reveals distinct diversification patterns among LysR-type transcriptional regulators in the ESKAPE pathogen Pseudomonas aeruginosa.

Pseudomonas aeruginosa, a harmful nosocomial pathogen associated with cystic fibrosis and burn wounds, encodes for a large number of LysR-type transcriptional regulator proteins. To understand how and why LTTR proteins evolved with such frequency and to establish whether any relationships exist within the distribution we set out to identify the patterns underpinning LTTR distribution in P. aeruginosa and to uncover cluster-based relationships within the pangenome. Comparative genomic studies revealed that in the JGI IMG database alone ~86 000 LTTRs are present across the sequenced genomes (n=699). They are widely distributed across the species, with core LTTRs present in >93 % of the genomes and accessory LTTRs present in <7 %. Analysis showed that subsets of core LTTRs can be classified as either variable (typically specific to P. aeruginosa) or conserved (and found to be distributed in other Pseudomonas species). Extending the analysis to the more extensive Pseudomonas database, PA14 rooted analysis confirmed the diversification patterns and revealed PqsR, the receptor for the Pseudomonas quinolone signal (PQS) and 2-heptyl-4-quinolone (HHQ) quorum-sensing signals, to be amongst the most variable in the dataset. Successful complementation of the PAO1 pqsR - mutant using representative variant pqsR sequences suggests a degree of structural promiscuity within the most variable of LTTRs, several of which play a prominent role in signalling and communication. These findings provide a new insight into the diversification of LTTR proteins within the P. aeruginosa species and suggests a functional significance to the cluster, conservation and distribution patterns identified.

Regioselective Partial Hydrogenation and Deuteration of Tetracyclic (Hetero)aromatic Systems Using a Simple Heterogeneous Catalyst.

The introduction of added '3-dimensionality' through late-stage functionalisation of extended (hetero)aromatic systems is a powerful synthetic approach. The abundance of starting materials and cross-coupling methodologies to access the precursors allows for highly diverse products. Subsequent selective partial reduction can alter the core structure in a manner of interest to medicinal chemists. Herein, we describe the precise, partial reduction of multicyclic heteroaromatic systems using a simple heterogeneous catalyst. The approach can be extended to introduce deuterium (again at late-stage). Excellent yields can be obtained using simple reaction conditions.

Regioselective Iridium-Catalyzed C8-H Borylation of 4-Quinolones via Transient O-Borylated Quinolines.

The quinolone-quinoline tautomerization is harnessed to effect the regioselective C8-borylation of biologically important 4-quinolones by using [Ir(OMe)(cod)]2 as the catalyst precursor, the silica-supported monodentate phosphine Si-SMAP as the ligand, and B2 pin2 as the boron source. Initially, O-borylation of the quinoline tautomer takes place. Critically, the newly formed 4-(pinBO)-quinolines then undergo N-directed selective Ir-catalyzed borylation at C8. Hydrolysis of the OBpin moiety on workup returns the system to the quinolone tautomer. The C8-borylated quinolines were converted to their corresponding potassium trifluoroborate (BF3 K) salts and to their C8-chlorinated quinolone derivatives. The two-step C-H borylation-chlorination reaction sequence resulted in various C8-Cl quinolones in good yields. Conversion to C8-OH-, C8-NH2 -, and C8-Ar-substituted quinolones was also feasible by using this methodology.

The Aldol-Tishchenko Reaction of Butanone, Cyclobutanone and a 3-Pentanone Derived Sulfinylimine and DFT Calculations of the Stereo-determining Step.

Herein, we present a highly diastereoselective method to furnish acyclic 3-amino-1,5-diol derivatives using a tandem double-aldol-Tishchenko protocol (dr up to >99 : 1) using a butanone derived sulfinylimine. In most cases only 1 diastereomer predominates, from a possible 16. The reaction is also regioselective. In addition, the highly challenging cyclobutanone and 3-pentanone derivatives are also amenable to a double-aldol-Tishchenko reaction, although the dr values are modest. Despite that, clean single diastereomers can be isolated, which should prove very useful in medicinal chemistry and other areas. Detailed DFT calculations support the observed stereoselectivities in all cases, providing a rationale for the excellent dr values in the butanone series and the moderate values for the 3-pentanone class.

Iridium-Catalyzed Borylation of 6-Fluoroquinolines: Access to 6-Fluoroquinolones.

The Ir-catalyzed C-H borylation of fluoroquinolines has been realized. The quinoline boronic ester formed undergoes a range of important transformations of relevance to medicinal chemistry. Judicious choice of the substituent at C4 on the quinoline facilitated the unmasking of a fluoroquinolone─the core structure of many antibiotics.

Pyrones Identified as LuxR Signal Molecules in Photorhabdus and Their Synthetic Analogues Can Alter Multicellular Phenotypic Behavior of Bacillus atropheaus.

Individual bacteria communicate by the release and interpretation of small molecules, a phenomenon known as quorum sensing (QS). We hypothesized that QS compounds extruded by Photorhabdus could be interpreted by Bacillus-a form of interspecies communication. We interrogate the structure-activity relationship within the recently discovered pyrone QS network and reveal the exquisite structural features required for targeted phenotypic behavior. The interruption of QS is an exciting, nonbiocidal approach to tackling infection, and understanding its nuances can only be achieved by studies such as this.

Stereoselective Installation of Five Contiguous Stereogenic Centers in a Double Aldol-Tishchenko Cascade and Evaluation of the Key Transition State through DFT Calculation.

The stereoselective formation of 5 contiguous chiral centers in a single pot reaction is demonstrated using an aldol, aldol-Tishchenko reaction of N-tert-butyl sulfinimines. One diastereoisomer (from 32 possibilities) predominates, and a series of cyclic and acyclic 3-amino-1,5-diol derivatives are synthesized in good yields (up to 80%) and excellent diastereoselectivities (up to >98:2 dr). Investigations support two reversible aldol steps, and multiple intermediates which are funnelled through a remarkably selective, irreversible, Tishchenko reduction, in a Curtin-Hammett phenomenon. DFT calculations using a disolvated (THF) model reveal the factors controlling stereoselectivity in the final irreversible Tishchenko step.

Mechanism and Origins of Stereoselectivity of the Aldol-Tishchenko Reaction of Sulfinimines.

Density functional theory computations have elucidated the mechanism and origins of stereoselectivity in McGlacken's aldol-Tishchenko reaction for the diastereoselective synthesis of 1,3-amino alcohols using Ellman's t-butylsulfinimines as chiral auxiliaries. Variations of stereochemical outcome are dependent on the nature of the ketone starting materials used, and the aspects leading to these differences have been rationalized. The intramolecular hydride transfer step is the rate- and stereochemistry-determining step, and all prior steps are reversible.

Detection of Pseudomonas aeruginosa quorum sensing molecules at an electrified liquid|liquid micro-interface through facilitated proton transfer.

Miniaturization of electrochemical detection methods for point-of-care-devices is ideal for their integration and use within healthcare environments. Simultaneously, the prolific pathogenic bacteria Pseudomonas aeruginosa poses a serious health risk to patients with compromised immune systems. Recognizing these two factors, a proof-of-concept electrochemical method employing a micro-interface between water and oil (w/o) held at the tip of a pulled borosilicate glass capillary is presented. This method targets small molecules produced by P. aeruginosa colonies as signalling factors that control colony growth in a pseudo-multicellular process known as quorum sensing (QS). The QS molecules of interest are 4-hydroxy-2-heptylquinoline (HHQ) and 2-heptyl-3,4-dihydroxyquinoline (PQS, Pseudomonas quinolone signal). Hydrophobic HHQ and PQS molecules, dissolved in the oil phase, were observed electrochemically to facilitate proton transfer across the w/o interface. This interfacial complexation can be exploited as a facile electrochemical detection method for P. aeruginosa and is advantageous as it does not depend on the redox activity of HHQ/PQS. Interestingly, the limit-of-linearity is reached as [H+] ≈ [ligand]. Density functional theory calculations were performed to determine the proton affinities and gas-phase basicities of HHQ/PQS, as well as elucidate the likely site of stepwise protonation within each molecule.

One-Pot, Tandem Wittig Hydrogenation: Formal C(sp3)-C(sp3) Bond Formation with Extensive Scope.

A one-pot, tandem Wittig hydrogenation of aldehydes with stabilized ylides is reported, representing a formal C(sp3)-C(sp3) bond construction. The tandem reaction operates under mild conditions, is high yielding, and is broad in scope. Chemoselectivity for olefin reduction is observed, and the methodology is demonstrated in the synthesis of lapatinib analogues and a formal synthesis of (±)-cuspareine. Early insights suggest that the chemoselectivity observed in the reduction step is due to partial poisoning of the catalyst, after step one, thus adding to the power of the one-pot procedure.

One-Pot Cross-Coupling/C-H Functionalization Reactions: Quinoline as a Substrate and Ligand through N-Pd Interaction.

Herein, we report a one-pot process that marries mechanistically distinct, traditional cross-coupling reactions with C-H functionalization using the same precatalyst. The reactions proceed in yields of up to 95%, in air, and require no extraneous ligand. The reactions are thought to be facilitated by harnessing the substrate quinoline as an N-ligand, and evidence of the palladium-quinoline interaction is provided by 1H-15N HMBC NMR spectroscopy and X-ray crystallographic structures. Application of the methodology is demonstrated by the quick formation of fluorescent, π-extended frameworks.

A structure-function analysis of interspecies antagonism by the 2-heptyl-4-alkyl-quinolone signal molecule from Pseudomonas aeruginosa.

In recent years, the alkyl-quinolone molecular framework has already provided a rich source of bioactivity for the development of novel anti-infective compounds. Based on the quorum-sensing signalling molecules 4-hydroxy-2-heptylquinoline (HHQ) and 3,4-dihydroxy-2-heptylquinoline (PQS) from the nosocomial pathogen Pseudomonas aeruginosa, modifications have been developed with markedly enhanced anti-biofilm bioactivity towards important fungal and bacterial pathogens, including Candida albicans and Aspergillus fumigatus. Here we show that antibacterial activity of HHQ against Vibrionaceae is species-specific and it requires an exquisite level of structural fidelity within the alkyl-quinolone molecular framework. Antibacterial activity was demonstrated against the serious human pathogens Vibrio vulnificus and Vibrio cholerae as well as a panel of bioluminescent squid symbiont Allivibrio fischeri isolates. In contrast, Vibrio parahaemolyticus growth and biofilm formation was unaffected in the presence of HHQ and all the structural variants tested. In general, modification to almost all of the molecule except the alkyl-chain end, led to loss of activity. This suggests that the bacteriostatic activity of HHQ requires the concerted action of the entire framework components. The only exception to this pattern was deuteration of HHQ at the C3 position. HHQ modified with a terminal alkene at the quinolone alkyl chain retained bacteriostatic activity and was also found to activate PqsR signalling comparable to the native agonist. The data from this integrated analysis provides novel insights into the structural flexibility underpinning the signalling activity of the complex alkyl-quinolone molecular communication system.

Electrochemical Sensing of Biotin Using Nafion-Modified Boron-Doped Diamond Electrode.

Nafion formed on the surface of a boron-doped diamond electrode allows for a chemosensing system for biotin. The modified electrode is capable of oxidizing biotin and offers a detection limit of 5 nM, the average normal level of biotin in blood plasma. The developed method was successfully applied to determine biotin in human plasma samples and a popular health product as two popular models.

Quinolones Modulate Ghrelin Receptor Signaling: Potential for a Novel Small Molecule Scaffold in the Treatment of Cachexia.

Cachexia is a metabolic wasting disorder characterized by progressive weight loss, muscle atrophy, fatigue, weakness, and appetite loss. Cachexia is associated with almost all major chronic illnesses including cancer, heart failure, obstructive pulmonary disease, and kidney disease and significantly impedes treatment outcome and therapy tolerance, reducing physical function and increasing mortality. Current cachexia treatments are limited and new pharmacological strategies are needed. Agonists for the growth hormone secretagogue (GHS-R1a), or ghrelin receptor, prospectively regulate the central regulation of appetite and growth hormone secretion, and therefore have tremendous potential as cachexia therapeutics. Non-peptide GHS-R1a agonists are of particular interest, especially given the high gastrointestinal degradation of peptide-based structures, including that of the endogenous ligand, ghrelin, which has a half-life of only 30 min. However, few compounds have been reported in the literature as non-peptide GHS-R1a agonists. In this paper, we investigate the in vitro potential of quinolone compounds to modulate the GHS-R1a in both transfected human cells and mouse hypothalamic cells. These chemically synthesized compounds demonstrate a promising potential as GHS-R1a agonists, shown by an increased intracellular calcium influx. Further studies are now warranted to substantiate and exploit the potential of these novel quinolone-based compounds as orexigenic therapeutics in conditions of cachexia and other metabolic and eating disorders.

The expanding horizon of alkyl quinolone signalling and communication in polycellular interactomes.

Population dynamics within natural ecosystems is underpinned by microbial diversity and the heterogeneity of host-microbe and microbe-microbe interactions. Small molecule signals that intersperse between species have been shown to govern many virulence-related processes in established and emerging pathogens. Understanding the capacity of microbes to decode diverse languages and adapt to the presence of 'non-self' cells will provide an important new direction to the understanding of the 'polycellular' interactome. Alkyl quinolones (AQs) have been described in the ESKAPE pathogen Pseudomonas aeruginosa, the primary agent associated with mortality in patients with cystic fibrosis and the third most prevalent nosocomial pathogen worldwide. The role of these molecules in governing the physiology and virulence of P. aeruginosa and other pathogens has received considerable attention, while a role in interspecies and interkingdom communication has recently emerged. Herein we discuss recent advances in our understanding of AQ signalling and communication in the context of microbe-microbe and microbe-host interactions. The integrated knowledge from these systems-based investigations will facilitate the development of new therapeutics based on the AQ framework that serves to disarm the pathogenesis of P. aeruginosa and competing pathogens.

Mechanistic studies on the palladium-catalyzed cross-dehydrogenative coupling of 4-phenoxy-2-coumarins: experimental and computational insights.

Delineating the mechanistic features of C-H activation in aryl-heteroaryl coupling is an important step in the design of selective, catalytic processes. Herein we use the intramolecular dehydrogenative coupling of 4-phenoxy-2-coumarins as a model study. Computational results and experimental studies suggest that CMD is operative in the cleavage of both C-H bonds, and that the heteroaryl C-H is cleaved initially.

Rapid Electrochemical Detection of Pseudomonas aeruginosa Signaling Molecules by Boron-Doped Diamond Electrode.

As the leading cause of morbidity and mortality of cystic fibrosis (CF) patients, early detection of Pseudomonas aeruginosa (PA) is critical in the clinical management of this pathogen. Herein, we describe rapid and sensitive electroanalytical methods using differential pulse voltammetry (DPV) at a boron-doped diamond (BDD) electrode for the detection of PA signaling biomolecules. Monitoring the production of key signaling molecules in bacterial cultures of P. aeruginosa PA14 over 8 h is described, involving sample pretreatment by liquid-liquid and solid-phase extraction. In addition, direct electrochemical detection approach of PA signaling molecules is also reported in conjunction with hexadecyltrimethylammonium bromide (CTAB) to disrupt the bacterial membrane.

Analogues of Pseudomonas aeruginosa signalling molecules to tackle infections.

The emergence of antibiotic resistance coupled with the lack of investment by pharmaceutical companies necessitates a new look at how we tackle bacterial infections. An intriguing tactic is the interruption of bacterial communication systems. This non-biocidal approach would circumvent the evolutionary pressure on bacteria to mutate and develop resistance. In many pathogenic microorganisms, communication systems, collectively termed quorum sensing (QS), have been observed to control a number of bacterial behaviours including expression of virulence factors and the development of biofilms. QS signalling molecules and their biomimetics, therefore, represent a rational target for the disruption of cooperative behaviour and thus the development of novel antimicrobial strategies. Herein we review recent developments towards the interference of Pseudomonas aeruginosa QS using signalling molecules and their mimetics.