SENSTIVE ENANTIOMERIC ANALYSIS OF N-ACYL HOMOSERINE LACTONES IN BACTERIAL MATRICES

Abstract

**Please note that the full text is embargoed until 12/13/2024** ABSTRACT: Communication between unicellular organism has long been studied with many facets yet to be discovered. A specific type of communication in gram-negative bacteria is quorum sensing (QS). N-acyl homoserine lactones (N-HLs) are the molecular signals in this process. At a critical concentration of N-HLs, all bacteria of the same population stop behaving like individuals and in a concerted manner induce a specific gene expression which ultimately benefits the entire population in major. This phenomenon has been coined with the term quorum sensing with the main signaling molecules dubbed L-N-acyl homoserine lactones (L-N-HL). Quorum sensing signaling pathways have their influence in a vast majority of realms, from nosocomial bacterial infections, to crop debilitation, from exchange of genetic code and food spoilage to microbial warfare and even symbiosis. For these reasons and more the study of bacterial signaling in quorum sensing has long been explored. It has however, long been disregarded the possibility of the production of the enantiomeric counterpart of these signaling molecules; D-N-acyl homoserine lactones (D-N-HL). For this reason, almost exclusively, has the analysis, detection and characterization of N-acyl homoserine lactones (N-HLs) been treated in an achiral fashion. To analyze these N-HLs in vivo another hurdle manifest as the complex biological and environmental matrices in which these signaling molecules are found. This dissertation aims to 1) develop proper, sensitive, selective and specific chiral chromatographic methods for the comprehensive analysis of D,L-N-HLs and their extraction from representative difficult matrices and 2) supply a myriad of studies in which these methods are a applied to bacterial matrices, displaying their efficacy the chiral analysis of both L- and D-N-HL in vivo. New methods involving high performance liquid chromatography and gas chromatography coupled to mass spectrometry (MS) were developed and evaluated for the chiral analysis of the three main classes of D,L-N-acyl homoserine lactones (N-HL). A derivatization method using N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) with 1% v/v trimethylchlorosilane (TMCS) was adapted for the analysis of the heat labile N-3-hydroxyacyl and N-3-oxoacyl homoserine lactones by gas chromatography. This method was used to analyze and detect L- and D-N-HL in bacterial cultures of B. cepacia and V. fischeri with limits of detection down to a ng/mL. CHIRALPAK IC-3s and an achiral stationary phase column were placed in tandem for the comprehensive analysis of all N-HL by LC. The first comprehensive chiral methods were developed for LC-MS/MS and GC-MS/MS for the analysis of trace-mounts of N-HL in bacterial matrices. Proper recovery studies on D,L-N-HL were performed by coupling to hydrophilic-lipophilic balanced solid phase extraction (HLB-SPE) phases. The utility of these HLB-SPE phases is their broad polarity selectivity as well as their robustness with aqueous samples. Percent recoveries for D,L-N-HL ranged from 80% to 105% recovery when using LC-MS/MS as well as obtaining method LODs to the level of a pg/mL. Using these simple, robust and comprehensive methods we were able to identify and quantify various L- and D-N-HL, that were expected as well as previously unreported, in bacterial matrices of Pseudomonas aeruginosa, Burkholderia cepacia, Pectobacterium atroscepticum and Vibrio fischeri.