Influence of the d/l configuration of N-acyl-homoserine lactones (AHLs) and analogues on their Lux-R dependent quorum sensing activity
Introduction
Bacterial Quorum Sensing (QS) is an intercellular communication system employed by numerous species of bacteria [1], [2], [3]. This process involves a coordination or repression of the genome based on chemical messengers, called autoinducers, which are synthesized by the LuxI protein family and interact with the LuxR protein family [3], [4], [5]. Many proteobacteria have been found to have a LuxR-AHL based QS response, with each having at least the same key components [3]. In Gram negative bacteria, autoinducers are related to acylated l-homoserine lactones (AHL) with diverse structural variations, such as different acyl chain lengths or the eventual presence of a 3-oxo or 3-hydroxy function. As a potential anti-bacterial strategy, the investigation of QS mimics represents an attractive alternative therapeutic approach for the treatment of human and plant bacterial infections. This is why the synthesis of small molecules which are capable of modulating bacterial QS systems has been widely studied in recent years [2], notably with experiments designed around native bacterial auto-inducers [2], [3], [6].
Stereochemistry is a key aspect of molecular recognition for biological systems. As such, receptors and enzymes are often highly stereospecific, only recognizing one stereoisomer of a ligand. The first discovery of the AHL signal was in the l-configuration, from a comparison of the c.d. spectra of the natural product and two synthetic isomers [7]. In 2004, D.R. Spring et al. reported that the l-isomer is probably reponsible for the QS autoinducing activity in P. aeruginosa and Serratia 39,006 when both enantiomers of its autoinducer were assayed [8], [9]. However, until now, there has been no systematic demonstration showing the stereochemical significance of QS autoinducers in various species of bacteria except for the few studies, cited above, pertaining to the investigation of the enantiomeric character of AHLs. In most cases, it was thought that probably only the l-isomers of AHL type autoinducers were responsible for the QS modulation and, consequently, only l-AHL analogues were considered for research without considering their d-isomers. For example, several families of racemic active analogues have been reported by our group, over several years, with variations in the side chain or in the amide mimics [10], [11], [12], [13], [14], [15], [16]. However, the ability of each pure enantiomer of these analogues, such as sulfonamides [12], ureas [11], sulfonylureas [14] and β-ketoamide AHL derivatives [17], to attenuate or induce QS modulation has never been studied in a systematic manner. With the aim of determining to what extent the d/l configuration of AHLs is a key parameter in the design of QS modulators, we report below our investigation into the influence of the d/l configuration of OHHL and some analogues, chosen from various agonsists or antagonists, on their ability to modulate QS.
In this study, we report our results on the synthesis and biological evaluation of a variety of optically pure AHLs and analogues, chosen from the QS-active compounds in our library and representative of several structural variations, namely OHHL, the natural ligand of Vibrio fischeri as l and d isomers, together with compounds 2–4. Compound 2 was described as an agonist and compounds 3–4 as antagonists when tested as a racemic mixture on the LuxR-regulated QS system.[10], [11], [12] All the compounds were prepared from l- and d-homoserine lactone hydrobromide via amidation with reagents. The enantiomeric purity was assessed by polarimetry, with complementary measurements from two methods, namely NMR, using an NMR shift reagent, and chiral HPLC. The bioassay was conducted in E. coli by measuring the level of inducing or inhibiting bioluminescence. In addition, the molecular modelling study of these enantiomers has also been investigated.
Section snippets
Synthesis
For the synthesis of AHLs, as previously described [18], l and d-methionine were alkylated with 2-bromoacetic acid, in acidic conditions, to give l- and d-homoserine lactone hydrobromide. These compunds were then acylated with the corresponding acylated Meldrum’s acid (d-isomers of OHHL, the L form was purchased) to yield optically pure β-ketoamide AHLs (Scheme 1). This method was based on an adapted DCC/DMAP-mediated coupling of carboxylic acid derivatives with Meldrum’s acid as the key
Conclusion
In conclusion, we have synthesized both optically pure enantiomers of AHLs and analogues, for which only racemic mixtures had been previously reported [10], [11], [12], [26], and evaluated their Lux-R dependent QS activity. The l-isomers are confirmed as being the only, or at least the most active, enantiomers. However, we found that for OHHL, and for the very similar AHL non-oxo analogue 2, the d-isomer should not be considered as totally inactive on QS, with EC50 values of 5 and 25 µM,
General
l-OHHL was purchased from Sigma-Aldrich. Optical rotations were measured on a Perkin Elmer 241 or Jasco P1010 polarimeter with a 10 cm cell (concentration c expressed as g/100 mL). HPLC was performed with a Perkin Elmer system equipped with a Flexar pump on a DAICEL Chiralpak AD column, and using UV (220 nm) as the detector (Flexar).
Procedure A:[12]
To a solution of l/d-Homoserine lactone hydrobromide, in 10 mL of dichloromethane, was added 1.2 equivalent of acyl chloride and 2.2 equivalent of triethylamine, at
Acknowledgements
Financial support from MESR (France), CNRS (France) and ANR “SENSOR” is gratefully acknowledged. S.Z.L. would like to thank the Chinese Scholarship Council for a Grant. The authors thank Valerie James for improving the English of the manuscript.
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