Synthesis and analysis of stable isotope-labelled N-acyl homoserine lactones

Virulence attenuation in pathogenic bacteria without the use of antibiotics reduces the threat of infection while limiting the evolution of multidrug resistant strains. In many bacteria, virulence is regulated by population density dependent genetic switches, a phenomenon known as quorum sensing (QS). QS depends on the synthesis, transmission, and perception of a variety of low molecular weight signal molecules generically classified as autoinducers. Gram‐negative bacteria, such as Pseudomonas aeruginosa, predominantly “communicate” population density using N‐acyl‐L‐homoserine lactones (AHLs). Crucial to developing Gram‐negative QS modulating strategies, as well as modeling the presence/absence of QS under “real‐world” conditions, is the ability to accurately estimate the relative levels of the ring‐closed (bioactive) and ring‐open (bio‐inactive) forms. While some AHLs have been studied for their hydrolysis (ring‐opening) rates, no quantitative and comprehensive model for degradation exists. Here, we employ 1H‐NMR to monitor the hydrolysis of various AHLs under standard laboratory conditions at fixed acidities to develop such a model. Our model provides valuable insight into the availability of bioactive AHLs at relevant pH conditions and is a valuable tool in the development of QS‐control strategies and evaluation of AHL focused chemical assays and studies.

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“…AHL Synthesis . AHLs were synthesized in accordance with previously established methods, and verified by 1 H‐NMR.…”

Section: Methodsmentioning

confidence: 99%

Abiotic Hydrolysis Kinetics of N‐Acyl‐L‐homoserine Lactones: Natural Silencing of Bacterial Quorum Sensing Signals

Ziegler¹,

Brown²,

Nesnas³

et al. 2019

Eur J Org Chem

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“…Briefly, even numbered N ‐hexanoyl‐homoserine lactone (C6) to N ‐tetradecanoyl‐homoserine lactone (C14) and N ‐3‐oxohexanoyl‐homoserine lactone (OXO6) to N ‐3‐oxotetradecanoyl‐homoserine lactone (OXO14) were synthesized according to Hodgkinson et al (2011) and synthesis of N ‐3‐hydroxyhexanoyl‐homoserine lactone (OH6) to N ‐3‐hydroxytetradecanoyl‐homoserine lactone (OH14), including N ‐3‐hydroxytridecanoyl‐homoserine lactone (OH13), was performed as described in Cao et al (1995). N ‐3‐oxononanoyl‐homoserine lactone (OXO9) was synthesized as described in Syrpas et al (2014) and deuterated standards N ‐[3‐ 2 H 2 ]‐octanoyl‐homoserine lactone (C8D) and N ‐[3‐ 2 H 2 ]‐dodecanoyl homoserine lactone (C12D) were prepared according to Ruysbergh et al (2016). As internal standards (ISTDs) C8D, C12D as well as OXO9 and OH13 were selected to cover all possible polarities.…”

Section: Materials and Proceduresmentioning

confidence: 99%

Sampling, separation, and quantification of N‐acyl homoserine lactones from marine intertidal sediments

Stock

Cirri

Nuwanthi

et al. 2021

Limnology & Ocean Methods

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N‐acyl homoserine lactones (AHLs) are molecules produced by many Gram‐negative bacteria as mediators of cell‐cell signaling in a mechanism known as quorum sensing (QS). QS is widespread in marine bacteria regulating diverse processes, such as virulence or excretion of polymers that mediate biofilm formation. Associated eukaryotes, such as microalgae, respond to these cues as well, leading to an intricate signaling network. To date, only very few studies attempted to measure AHL concentrations in phototrophic microbial communities, which are hot spots for bacteria‐bacteria as well as microalgae‐bacteria interactions. AHL quantification in environmental samples is challenging and requires a robust and reproducible sampling strategy. However, knowing about AHL concentrations opens up multiple perspectives from answering fundamental ecological questions to deriving guidelines for manipulation and control of biofilms. Here, we present a method for sampling and AHL identification and quantification from marine intertidal sediments. The use of contact cores for sediment sampling ensures reproducible sample surface area and volume at each location. Flash‐freezing of the samples with liquid nitrogen prevents enzymatic AHL degradation between sampling and extraction. After solvent extraction, samples were analyzed with an ultra‐high performance liquid chromatography‐high resolution mass spectrometry (UHPLC‐HRMS) method that allows to baseline‐separate 16 different AHLs in less than 10 min. The sensitivity of the method is sufficient for detection and quantification of AHLs in environmental samples of less than 16 cm3.

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“…AHL synthesis : Following the procedure of Ruysbergh [59] et al. for synthesizing native AHLs with slight modification, compounds 1 , 2 , and 4 were prepared by first dissolving freshly synthesized homoserine lactone hydrochloride (275 mg, 2 mmol) in deionized water (15 mL) and treating the resulting solution with the corresponding carboxylic acid (2 mmol), 1‐ethyl‐3‐(3‐dimethylaminopropyl)carbodiimide (380 mg, 2 mmol, 1 equiv.…”

Section: Methodsmentioning

confidence: 99%

β‐Cyclodextrin Encapsulation of Synthetic AHLs: Drug Delivery Implications and Quorum‐Quenching Exploits

et al. 2020

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Many bacteria, such as Pseudomonas aeruginosa, regulate phenotypic switching in a population density‐dependent manner through a phenomenon known as quorum sensing (QS). For Gram‐negative bacteria, QS relies on the synthesis, transmission, and perception of low‐molecular‐weight signal molecules that are predominantly N‐acyl‐l‐homoserine lactones (AHLs). Efforts to disrupt AHL‐mediated QS have largely focused on the development of synthetic AHL analogues (SAHLAs) that are structurally similar to native AHLs. However, like AHLs, these molecules tend to be hydrophobic and are poorly soluble under aqueous conditions. Water‐soluble macrocycles, such as cyclodextrins (CDs), that encapsulate hydrophobic guests have long been used by both the agricultural and pharmaceutical industries to overcome the solubility issues associated with hydrophobic compounds of interest. Conveniently, CDs have also demonstrated anti‐AHL‐mediated QS effects. Here, using fluorescence spectroscopy, NMR spectrometry, and mass spectrometry, we evaluate the affinity of SAHLAs, as well as their hydrolysis products, for β‐CD inclusion. We also evaluated the ability of these complexes to inhibit wild‐type P. aeruginosa virulence in a Caenorhabditis elegans host infection study, for the first time. Our efforts confirm the potential of β‐CDs for the improved delivery of SAHLAs at the host/microbial interface, expanding the utility of this approach as a strategy for probing and controlling QS.

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Synthesis and analysis of stable isotope-labelled N-acyl homoserine lactones

Cited by 10 publications

References 59 publications

Abiotic Hydrolysis Kinetics of N‐Acyl‐L‐homoserine Lactones: Natural Silencing of Bacterial Quorum Sensing Signals

Abiotic Hydrolysis Kinetics of N‐Acyl‐L‐homoserine Lactones: Natural Silencing of Bacterial Quorum Sensing Signals

Sampling, separation, and quantification of N‐acyl homoserine lactones from marine intertidal sediments

β‐Cyclodextrin Encapsulation of Synthetic AHLs: Drug Delivery Implications and Quorum‐Quenching Exploits

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